1. Technical Field
This disclosure relates to a method and system for performing a serological agglutination assay in a liquid sample. The system provides a simple method for creating an in-situ sample/reagent admixture within a sample analysis chamber without the use of any precision fluid-handling components. The relative and absolute concentrations of the reactants may be ascertained in any small area of the reaction vessel.
2. Background Information
In most assays it is necessary to provide an exact dilution of the sample to be analyzed so that the concentration of the analyte can be brought into the useful range of the assay, and since this dilution affects the concentration of the analyte, the precision and accuracy of the test to a large extent depends upon the precision and accuracy of the dilution. One reason for this dilution is that immunoassays are affected by a phenomenon known as the prozone effect. The term “prozone” as used in this disclosure shall refer to conditions of antibody excess where generally in precipitation or agglutination-based immunoassays reactions are inhibited or prevented, the postzone, where conditions of antigen excess in an immunoassay where agglutination or precipitation reactions are inhibited, and the “hook effect” where conditions of antigen excess result in falsely low results. Conditions where the prozone effects occur can result in false negatives and falsely low results with catastrophic results to the patient.
Each assay combination has an empirically defined working range and assays must be performed with samples and reactants in the appropriate dilutions. This type of dilution has traditionally been accomplished through the use of precision fluid-handling components or manual repeating of the assay at higher dilutions of the antibody to see if the negative is a true negative. Although these can be very accurate, they require careful calibration and greatly add to the complexity of automated instrumentation. Additionally the range of analyte present in the sample may exceed the dynamic range of the assay and may require further dilution of the sample for accurate results. Additionally, the prior art requires many chambers to contain the various concentrations of reactants.
Serologic assays, such as for antibodies to infectious disease pathogens, are important in that they tell of either existing immunity due to immunization or to previous or current exposure, depending on the class of immunoglobulin present, to the infectious agent. Similarly, they may be used to detect auto-immunity and the like. There are a number of assay types performed, including agglutination, complement-fixation, precipitation, etc. One almost universal feature of such tests is the need to dilute the sample a number of times in order to detect the point where the antibodies are no longer effective to cause a positive test. This is referred to as the “titer”, the titer being the highest dilution of the patient's serum or plasma that yields detectable agglutination or measured reaction with the test antigen. This, in effect, requires the performance of many separate tests in separate chambers to arrive at the result. Another problem with such assays is that the end-points are sometimes difficult to determine, thus adding a significant error to the titer determination. Automation can increase the test efficiency and accuracy, but performing the dilutions by an instrument is very difficult and time consuming including the need to first define the desired dilution which can vary from test to test and the multiple dilution steps are very complex.
It would be desirable to provide a method and apparatus for measuring antibody titers in an automated system which does not require multiple dilutions and that removes the risk of false negatives due to the prozone effect.